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Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.

The platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) interacts with podoplanin on the surface of certain types of tumor cells, and this interaction facilitates tumor metastasis. CLEC-2 is also involved in thrombus formation and its stabilization. Because CLEC-2–depleted mice are protected from experimental lung metastasis and thrombus formation and do not show increased bleeding time, CLEC-2 may serve as a good target for antimetastatic or antithrombotic drugs. We screened 6770 compounds for their capability to inhibit CLEC-2–podoplanin binding using an enzyme-linked immunosorbent assay. In the first screening round, 63 compounds were identified and further evaluated by flow cytometry using CLEC-2–expressing cells. We identified protoporphyrin IX (H2-PP) as the most potent inhibitor and modified its hematoporphyrin moiety to be complexed with cobalt (cobalt hematoporphyrin [Co-HP]), which resulted in an inhibitory potency much stronger than that of H2-PP. Surface plasmon resonance analysis and molecular docking study showed that Co-HP binds directly to CLEC-2 at N120, N210, and K211, previously unknown podoplanin-binding sites; this binding was confirmed by analysis of CLEC-2 mutants with alterations in N120 and/or K211. Co-HP at a concentration of 1.53 μM inhibited platelet aggregation mediated through CLEC-2, but not that mediated through other receptors. IV administration of Co-HP to mice significantly inhibited hematogenous metastasis of podoplanin-expressing B16F10 cells to the lung as well as in vivo arterial and venous thrombosis, without a significant increase in tail-bleeding time. Thus, Co-HP may be a promising molecule for antimetastatic and antiplatelet treatment that does not cause bleeding tendency.

Bisphenol A (BPA) is an artificial environmental endocrine disrupter. Excess exposure to BPA may induce many disorders in the metabolism and cardiovascular system. However, the underlying toxicological mechanisms remain largely unknown. In this study, we administered genetically hyperlipidemic Watanabe heritable hyperlipidemic (WHHL-MI) rabbits (male, 14 week old), which have more common features with humans than the mouse and rat especially in the metabolism and cardiovascular system, with BPA at 40 mg kg–1 day–1 for 8 weeks by gavage and compared their plasma lipids, glucose and insulin response with those of the vehicle group. All of the rabbits were sacrificed, and their pancreas, liver, adipose tissue, heart and aorta were analyzed using histological and morphometric methods. Furthermore, we treated human hepatoma HepG2 cells and human umbilical cord vein endothelial cells (HUVECs), with different doses of BPA based on the serum BPA levels in the WHHL rabbits for 6 h to investigate the possible molecular mechanisms. Our results showed that BPA-treated rabbits showed insulin resistance, prominent adipose accumulation and hepatic steatosis. Additionally, BPA exposure also caused myocardial injury and enhanced the development of atherosclerosis in the aortic arch with increased macrophage number (86%) and advanced lesion areas (69%). Increased expression of inflammatory genes found in the liver of BPA-treated rabbits along with the up-regulation of ER stress, lipid and glucose homeostasis and inflammatory genes in the cultured HepG2 cells and HUVECs suggest that BPA may induce metabolic disorders and enhance atherosclerosis through regulating above molecular pathways in the liver and endothelium. Copyright © 2015 John Wiley & Sons, Ltd.

Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and β-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. Summary Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and β-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.

In-stent thrombosis (IST) after carotid artery stenting (CAS) is a rare but potentially devastating complication. We present a case of early IST after CAS despite sufficient antiplatelet therapy in a patient with bladder cancer. A 77-year-old man under preventive triple antiplatelet therapy underwent CAS without any intra- or periprocedural complications. However, the patient developed a large asymptomatic IST 6 days after CAS. Anticoagulant therapy with argatroban was reintroduced to treat IST concomitant with antiplatelet agents. Subsequently, the IST shrank and disappeared without any thrombotic symptoms. Malignancy is regarded as an acquired thrombophilic condition associated with a significant risk of thrombosis. In the field of coronary stents, cancer is associated with a significant increasing risk of IST. The cause of IST in our case was possibly related in hypercoagulable state because of the patient's cancer. Attention for IST should be paid in CAS cases with these risk factors, and repeated examination is recommended.

It is implicated that diabetic patients are more resistant to aspirin therapy than patients with other diseases or healthy individuals. We evaluated the inhibitory effects of aspirin on aggregation and the cyclooxygenase activity of platelets of 10 patients with severe type-2 diabetes mellitis (DM) and compared the results with those of healthy individuals. Although platelet aggregation had a tendency to be more resistant to aspirin with the DM group, there was no significant difference in half maximal inhibitory concentration 50 values of aspirin on the cyclooxygenase activity between the patients with DM and healthy individuals. Thus, the residual platelet aggregability uninhibited by aspirin appears to be independent of the cyclooxygenase activity. Since adenosine diphosphate (ADP) receptor blocking almost completely inhibited the residual platelet aggregability, it is suggested that hyperreactivity to ADP is more prevalent in patients with DM.

Essentials The role of C-type lectin-like receptor-2 (CLEC-2) in cancer progression is unclear. CLEC-2-depleted mouse model is generated by using a rat anti-mouse CLEC-2 monoclonal antibody. CLEC-2 depletion inhibits hematogenous tumor metastasis of podoplanin-expressing B16F10 cells. CLEC-2 depletion prolongs cancer survival by suppressing thrombosis and inflammation. SummaryBackground C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2–podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.

Introduction Cilostazol has been shown to be effective for prevention and treatment of cerebral infarction. However, there appears to be no widely accepted method appropriate for monitoring cilostazol. We attempted to establish an assay system for cilostazol monitoring, using platelet aggregation induced by arachidonic acid (AA) in the presence of PGE1 which upregulates intracellular cyclic AMP. Methods Blood was drawn from stroke patients before and after cilostazol intake. AA-induced platelet aggregation after pretreatment with 0 ~ 30 nM PGE1 for 2 minutes was measured by light transmittance aggregometry. Results AA-induced platelet aggregation was 73.1 ± 2.2% in the absence of PGE1, and pretreatment with 30 nM PGE1 had virtually no inhibitory effect on platelet aggregation prior to cilostazol intake. In contrast, after cilostazol intake, 30 nM PGE1 significantly inhibited platelet aggregation to 12.7 ± 4.5% (p = 7.8 × 10(− 11)) , while in the absence of PGE1 platelet aggregation remained similar to that of prior-to-cilostazol value (70.6 ± 3.5%). The plasma concentration of cilostazol ranged from 0.55 to 3.51 μM. In the presence of 30 nM PGE1, all the patients with cilostazol concentrations exceeding 1 μM had their platelet aggregation inhibited almost completely. ROC analysis suggests that AA-induced platelet aggregation in the presence of 30 nM PGE1 had the excellent sensitivity (90.5%) and specificity (88.4%) for monitoring cilostazol. Conclusions AA-induced platelet aggregation in the presence of 30 nM PGE1 could give good estimate on plasma concentrations of cilostazol. It is suggested that this system is a good tool for monitoring cilostazol.

We retrospectively studied 89 patients with chronic hepatitis C virus (HCV) infection, including 50 chronic hepatitis (CH) cases, 18 liver cirrhosis (LC) cases, and 21 LC with hepatocellular carcinoma (LC + HCC) cases, with regard to various factors related with thrombocytopenia. The platelet count decreased with the stage advancement of liver diseases. Multiple regression analysis revealed that splenomegaly and von Willebrand factor (vWF) were explanatory variables that correlated with thrombocytopenia. Splenomegaly appears to be the most responsible factor, although there are a considerable number of thrombocytopenic cases without splenomegaly, suggesting other factors may also be responsible. The vWF level is inversely correlated with the platelet count. Soluble thrombomodulin, a marker of endothelial dysfunction, increases with the advancement of liver fibrosis. It is positively correlated with vWF and inversely with the platelet count. Our present results imply that vascular endothelial dysfunction is also involved in thrombocytopenia during chronic HCV infection.

A simple, validated method to measure platelet function is unavailable for bedside use. Measurement of platelet retention rate using a column of collagen-coated beads and whole blood is a new, simple assay that reflects platelet aggregation. This study was aimed to examine the utility of this assay to assess efficacy of antiplatelet drug therapy.Citrated whole blood (1.5 ml) in a syringe was passed through a polyvinyl tube packed with collagen-coated beads for 40 seconds using a syringe pump. Platelet retention rate in the column was calculated from platelet counts in blood before and after passage. An increase in the retention rate reflects an increase in platelet activity. This new platelet retention assay and the traditional optical aggregometry assay were performed in 331 patients with stable coronary artery disease (CAD).The retention rate was significantly reduced in patients taking dual antiplatelet therapy (aspirin plus clopidogrel or ticlopidine) compared with aspirin alone. There was a significant linear correlation between the platelet retention rate and platelet aggregability measured by the traditional method (r=0.44, p0.001). In multivariate Cox proportional hazards analysis, higher platelet retention rate was an independent predictor of future cardiovascular events in patients on dual antiplatelet therapy (hazard ratio 3.9, 95% CI 1.6 to 9.5, p=0.003).Measurement of the platelet retention rate in a column of collagen-coated beads may be useful for monitoring the efficacy of antiplatelet drug therapy in patients with CAD.

Platelet aggregometry by the laser light scattering (LS) method is sufficiently sensitive to detect small platelet aggregates that form spontaneously in vitro in the absence of agonists. Platelet aggregation without agonists is named spontaneous platelet aggregation (SPA). Since SPA has been suggested to be associated with various thrombotic diseases, it is essential to measure SPA and to establish a standard range of SPA values. In this study, we measured SPA in 167 healthy subjects by the LS method and attempted to clarify various factors influencing SPA, including the blood collection procedure. We also attempted to establish a tentative standard range of SPA values. SPA was quantitatively measured in terms of the maximum total LS intensity, which reflects small aggregates formed over 10 minutes (SMAX) and the area under the total LS intensity curve of small aggregates (SAUC). Since both the values of SMAX and SAUC were skewed and the log SMAX and log AUC values showed a normal distribution, the statistical analyses were performed using log SMAX and log SAUC. The log SMAX and log SAUC were significantly higher in the samples collected using a tourniquet and/or a 21 G needle, than in those collected without a tourniquet and/or with an 18 G needle. The log SAUC values were significantly lower in samples obtained with a syringe and/or 3.8% sodium citrate than in those obtained in vacuum sampling tubes and/or 3.13% or 3.14% sodium citrate. The Ht and plasma glucose concentration influenced the log SMAX values. We propose that to standardize SPA measurements, the measurements should be completed within two hours of blood sample collection and collected using the regular concentration of citrate. The standard range of SMAX values measured in samples obtained using a tourniquet and a 21 G needle was 2.0-23.99 (*10(3) mV*count). The standard range of SAUC values measured under same conditions was 0.58-9.12 (*10(6) mV*count*min). The standard range of SMAX values measured in samples obtained using a tourniquet, 21 G needle and a vacuum tube was 1.7-29.51 (*10(3) mV*count). The standard range of SAUC values measured under same conditions was 0.59-9.33 (*10(6) mV*count*min).

Summary. Background: Glycoprotein (GP) Ib, a platelet von Willebrand factor (VWF) receptor, plays a crucial role in thrombosis and hemostasis. As recent reports have suggested that GPIb partially locates in a particular region, designated as glycosphingolipid-enriched microdomains (GEMs), we hypothesized that GEMs play a central role in GPIb-mediated platelet activation. Methods: Platelets were stimulated by VWF/botrocetin to activate platelets through GPIb. GEMs and non-GEMs were isolated by sucrose density gradient ultracentrifugation and the location of signaling molecules characterized. The role of GEMs-mediated signaling in platelet behavior was tested by platelet aggregation and by platelet interaction with immobilized VWF under flow conditions when GEMs were disrupted by methyl-β-cyclodextrin (MβCD). Results: GPIb was partially translocated to GEMs upon VWF/botrocetin stimulation. Immunoprecipitation of GPIb in GEMs and non-GEMs revealed that the tyrosine kinases, Src and Lyn, were associated with GPIb only in GEMs after GPIb-stimulation, and not in non-GEMs. Activation of PLCγ2 was more intense in GEMs than non-GEMs. Disruption of GEMs by MβCD strongly inhibited tyrosine phosphorylation of Syk and PLCγ2. Functional studies revealed that stable adhesion of platelets to a VWF-coated surface under flow was impaired by GEM disruption by MβCD. Conclusion: The combined results suggest that GEMs play an important role in GPIb-mediated platelet activation.

The inhibitory effects of ticlopidine on platelet function were evaluated in 18 healthy male volunteers using three different platelet function tests. Three methods include the recently developed small collagen-beads method, which evaluates the platelet response in whole blood samples under shear stress conditions, the conventional platelet aggregometry and a cone-plate viscometer which measures shear-induced platelet aggregation (SIPA). The latter two methods use platelet-rich plasma as measurement samples instead of whole blood. SIPA was significantly inhibited by the oral intake of ticlopidine. The conventional platelet aggregometry detected significant inhibition of ticlopidine on ADP-induced platelet aggregation. In contrast, ticlopidine moderately inhibited platelet aggregation induced by low concentrations of collagen, but not by high concentrations of collagen. With the collagen-bead column method, ticlopidine significantly inhibited platelet retention rates when the retention rates exceeded 30% prior to ticlopidine uptake. On the other hand, there was no significant inhibition when the original retention rates prior to ticlopidine uptake were below 30%. The three methods all proved useful to evaluate the effect of ticlopidine on platelet function. However, taking into consideration easy procedures, lower costs and use of whole blood samples under shear stress conditions, we suggest the collagen-bead column can serve as an appropriate method for monitoring ticlopidine therapy.

Background and Purpose: It is widely accepted that antiplatelet therapy is effective for secondary prevention of atherosclerotic vascular diseases. We performed a double-blind, controlled clinical-pharmacological study to investigate the antiplatelet efficacy of sarpogrelate, a selective 5-hydroxytryptamine (5-HT2A) receptor antagonist, in patients with ischemic stroke, using a new assessment system employing combinations of 5-HT and epinephrine as agonists. Methods: Forty-seven patients with ischemic stroke were randomly assigned to three groups: 15 patients received 25 mg sarpogrelate (group L), 16 patients received 50 mg (group M), and 15 patients received 100 mg (group H) orally, three times daily for 7 days. The effect was expressed as maximum intensity of platelet aggregation on the last day of medication. Two combinations of agonists, 0.5 µmol/l 5-HT plus 3 µmol/l epinephrine, and 1 µmol/l 5-HT plus 3 µmol/l epinephrine, were used to induce platelet aggregation. Results: With both combinations of agonists, sarpogrelate treatment inhibited platelet aggregation dose-dependently (p < 0.025, Jonckheere test). In multiple-group comparison, the effect in group H was greater than that in group L or M (p < 0.025, Wilcoxon rank-sum test). Conclusion: Sarpogrelate treatment inhibited platelet aggregation dose-dependently in patients with ischemic stroke, as judged by a new assessment system employing combinations of 5-HT and epinephrine as agonists.

Hot water extracts of the medicinal mushroom Agaricus brasiliensis were investigated for their inhibition of platelet aggregation. The extracts significantly inhibited human platelet aggregation induced by adenosine 5'-diphosphate (ADP), but not by collagen or thrombin receptor-activating peptide. The extracts also had a significant inhibitory effect on shape change and intracellular calcium mobilization induced by ADP via inhibition of ADP binding to the P2Y1 receptor. In addition, oral administration of the extracts resulted in prolonged tail bleeding time in mice. The marked antiplatelet activity of the mushroom extracts involving the P2Y1 receptor suggests their potential therapeutic use against vascular disorders.

We previously reported that platelet retention rates as measured with collagen-coated bead columns (the conventional column) reflect the processes of platelet adhesion and aggregation under low shear stress, and that this system could serve as an easy-to-use platelet aggregometry. With this column, platelet glycoprotein (GP) VI and GPIIb/IIIa, but not the GPIb-von Willebrand factor (VWF) interaction, play major roles in platelet activation. To develop a system that can better reflect the GPIb-VWF interaction under high shear stress, we designed a column containing small-sized beads (125-212 microm) coated with porcine collagen type I. As expected, the GPIb-VWF interaction played a crucial role in platelet retention rates at higher flow rates. Adenosine 5'-diphosphate, but not thromboxane A2, appears to support platelet activation in this system. The platelet retention rates among healthy individuals with the new columns are in the range wider than the conventional columns, and this diversity could be attributed to the broad range of the VWF antigen and/or its activity. It is suggested that this new column can serve as an easy-to-use method for evaluating the VWF antigen levels and its activity and for monitoring patients with thrombotic or bleeding disorders related to the VWF-GPIb interaction.

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets and plays an important role in vascular biology. In this study, we investigated Sph-1-P-related metabolism in the blood vessel, mainly using radio-labeled Sph and Sph-1-P. Sph was metabolically stable in the plasma, while it was converted into Sph-1-P in the presence of activated platelets. When the mixture of Sph-1-P and plasma was fractionated on a gel-filtration column, all Sph-1-P co-eluted with protein fractions that coincide with lipoproteins and albumin by agarose gel electrophoresis. When evaluated by polyacrylamide gel electrophoresis, 7.2 +/- 3.8%, 53.3 +/- 6.4%, and 39.5 +/- 7.9% of the radioactivity of Sph-1-P added to plasma was recovered in the low-density lipoprotein (LDL), high-density lipoprotein (HDL), and albumin fractions, respectively. On the other hand, 5.2 +/- 3.2%, 38.4 +/- 5.5%, and 56.3 +/- 5.7% of the radioactivity of Sph-1-P converted from Sph in collagen-stimulated platelets and released into the plasma was recovered in the LDL, HDL, and albumin fractions, respectively. When Sph-1-P release from activated platelets was examined, a stronger response was observed in the presence of albumin than lipoproteins, suggesting efficient Sph-1-P extraction from platelets by albumin. Finally, Sph-1-P, which is stable in the plasma, was markedly degraded by an ectophosphatase activity in the presence of vascular endothelial cells or in whole blood. Although Sph-1-P is stable in the plasma, it is likely that the level of this bioactive lipid is dynamically controlled by various factors including release from platelets, distribution among plasma proteins, and degradation by ectophosphatase.

Although the glass-bead column has been used to measure platelet adhesion, whether platelet interaction with glass beads represents physiologic processes remains unsettled. In an attempt to obtain more physiologic platelet responses, plastic beads coated with type I collagen have been recently developed to replace glass beads. In this study, we analyzed the factors responsible for platelet retention in the collagen-coated-bead column and investigated its possible clinical applications. We pumped citrated whole-blood samples into columns at a fixed speed with an injection pump and calculated platelet-retention rates by measuring platelet counts before and after passage through the columns. The platelet-retention rates, which were highly reproducible with samples from healthy donors, were reduced in a patient with glycoprotein (GP) VI deficiency but not in patients with type III von Willebrand disease. Anti-GPIIb/IIIa antibody and GRGDS peptide markedly inhibited platelet retention, whereas inhibition of the GPIb-von Willebrand factor or GPIa/IIa-collagen interaction had no effect. Data on the effects of various antiplatelet agents (including the antithrombin agent argatroban, prostacyclin, acetylsalicylic acid, and the ADP scavenger creatine phosphate/creatine phosphokinase) support the usefulness of this assay method in clinical application. Our findings suggest that GPVI and GPIIb/IIIa but not the GPIb-von Willebrand factor interaction are mainly involved in platelet retention in this column.

The binding of von Willebrand factor (VWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3– kinase (PI 3–kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3–kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3–kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)–fusion protein coprecipitated the p85 subunit of PI 3–kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3–kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3– kinase.

Background Although cross-talks between platelets and other blood cells are important in vivo, laboratory platelet aggregation tests have been performed mainly with the use of platelet-rich plasma (PRP) as samples. Methods that enable an efficient and sensitive detection of platelet aggregates in whole blood are being developed. Methods A flow cytometer equipped with an imaging device, the flow imaging cytometer 2 (FIC2), was used to detect platelet aggregates in whole blood. Results The FIC2 provides a resolution that is high enough to differentiate platelet aggregates from single platelets or other blood cells. Epinephrine elicited platelet aggregate formation in hirudin plus argatroban-treated whole blood, but not in PRP. The reconstitution study revealed that a small amount of adenosine diphosphate (ADP) from erythrocytes may play an important role in epinephrine-induced platelet aggregation (in whole blood), through mediation of P2Y1 receptors. When the inhibitory effect of beraprost, an antiplatelet agent, on platelet aggregation was assessed, analysis of whole blood samples with FIC2 proved to be the most sensitive among the methods available. Conclusions FIC2 is a promising device for detection of platelet aggregates in whole blood, with wide basic and clinical applications. Cytometry 48:194–201, 2002. © 2002 Wiley-Liss, Inc.

Bisphenol A (BPA) is an artificial environmental endocrine disrupter. Excess exposure to BPA may induce many disorders in the metabolism and cardiovascular system. However, the underlying toxicological mechanisms remain largely unknown. In this study, we administered genetically hyperlipidemic Watanabe heritable hyperlipidemic (WHHL-MI) rabbits (male, 14 week old), which have more common features with humans than the mouse and rat especially in the metabolism and cardiovascular system, with BPA at 40 mg kg(-1) day(-1) for 8 weeks by gavage and compared their plasma lipids, glucose and insulin response with those of the vehicle group. All of the rabbits were sacrificed, and their pancreas, liver, adipose tissue, heart and aorta were analyzed using histological and morphometric methods. Furthermore, we treated human hepatoma HepG2 cells and human umbilical cord vein endothelial cells (HUVECs), with different doses of BPA based on the serum BPA levels in the WHHL rabbits for 6 h to investigate the possible molecular mechanisms. Our results showed that BPA-treated rabbits showed insulin resistance, prominent adipose accumulation and hepatic steatosis. Additionally, BPA exposure also caused myocardial injury and enhanced the development of atherosclerosis in the aortic arch with increased macrophage number (86%) and advanced lesion areas (69%). Increased expression of inflammatory genes found in the liver of BPA-treated rabbits along with the up-regulation of ER stress, lipid and glucose homeostasis and inflammatory genes in the cultured HepG2 cells and HUVECs suggest that BPA may induce metabolic disorders and enhance atherosclerosis through regulating above molecular pathways in the liver and endothelium.

In this study, the receptors and signals involved in collagen-induced platelet spreading were examined. It was found that platelet spreading on collagen (presenting a polygon shape with a number of filopodialike projections) was inhibited by the anti-integrin alpha(2) antibody, suggesting the involvement of integrin alpha(2)beta(1) in this process. Studies with a glutathione-S-transferase fusion protein that binds specifically to activated Rac and in vitro p21-activated kinase (PAK) kinase assays revealed that Rac and PAK were activated during this collagen-activated process. Platelet spreading on collagen-coated surfaces was inhibited strongly by PP1 (a Src family kinase inhibitor) or weakly by wortmannin (a phosphatidylinositol 3-kinase [PI3-kinase] inhibitor) but not at all by Y-27632 (a Rho kinase inhibitor). The surfaces coated with anti-integrin alpha(2)beta(1) antibodies also induced platelet spreading (presenting an almost complete round shape) and activation of Rac and PAK, although more slowly than collagen-coated surfaces. The antibody-induced responses were strongly inhibited by PP1 or wortmannin but not by Y-27632. The same concentration of Y-27632 inhibited collagen-induced shape change of platelets in suspension. These findings suggest that Rac and/or PAK activation, but not Rho, may play certain roles in platelet spreading via integrin alpha(2)beta(1) and that Src family kinases and PI3-kinase participate in these processes. Furthermore, the difference between spreading on collagen and the anti-integrin antibody suggests the involvement of other receptor(s) (in addition to the integrin alpha(2)beta(1)) for collagen-induced spreading, the most likely candidate being glycoprotein VI.

Interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) stimulates tyrosine kinases and subsequent tyrosine phosphorylation events in human platelets. This study found that the combination of vWF and botrocetin, by interacting with GPIb, induced tyrosine phosphorylation of Fc receptor gamma-chain (FcR gamma-chain), Syk, linker for activation of T cells (LAT), and phospholipase C gamma2 (PLCgamma2). Pretreatment of platelets with 10 microM PP1 completely inhibited these tyrosine phosphorylation events. On GPIb stimulation, Src and Lyn formed a complex with FcR gamma-chain and Syk, suggesting that Src and Lyn are involved in FcR gamma-chain tyrosine phosphorylation and downstream signals. In spite of the PLCgamma2 tyrosine phosphorylation, however, there was no intracellular calcium release and inositol 1,4,5-trisphosphate production. In Brij 35 lysates, FcR gamma-chain was found to constitutively associate with GPIb. The number of GPIb expressed on FcR gamma-chain-deficient platelets was comparable to that of the wild-type, as assessed by flow cytometry. However, tyrosine phosphorylation of Syk, LAT, and PLCgamma2 in response to vWF plus botrocetin was significantly suppressed, suggesting that FcR gamma-chain mediates activation signals related to GPIb. Compared with the aggregation response of wild-type platelets, that of FcR gamma-chain-deficient platelets in response to vWF plus botrocetin was impaired, implying that FcR gamma-chain is required for the full activation of platelets mediated by GPIb. (Blood. 2001;97:3836-3845)

Although communications between platelets and endothelial cells or other blood cells are important in in vivo thrombus formation, laboratory platelet function tests are usually performed in isolation from these surrounding cells. In this study, we evaluated the effect of an antiplatelet drug, ibudilast (3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine), on platelet aggregation in the presence and absence of human umbilical vein endothelial cells (HUVECs) and with the use of platelet-rich plasma (PRP) or whole blood as platelet samples. Stimulation-dependent platelet aggregation was weakened in the presence of HUVECs, which was especially prominent when the thrombin receptor-activating peptide SFLL (compared with ADP and epinephrine) was used as an aggregating agent. Ibudilast hardly affected SFLL-induced platelet aggregation (in PRP), while this antiplatelet agent was found to clearly inhibit this SFLL-induced response in a concentration-dependent manner, in the presence of HUVECs. Ibudilast tended to inhibit ADP- or epinephrine-induced platelet aggregation in the presence of HUVECs, but the effects were not statistically significant. Enhanced inhibition by ibudilast of SFLL-induced platelet aggregation (in the presence of HUVECs) was reproduced with the use of whole blood samples when a screen filtration pressure method was employed. It is suggested that the platelet aggregation studies in the presence of endothelial cells and/or other blood cells provide us with valuable information on platelet reactivity in vivo and improvement of antiplatelet therapy.

Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxillin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxillin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A(2) analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin alphaIIbbeta3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2 (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on alphaIIbbeta3-mediated aggregation. Finally, on stimulation with PMA, Hic-5, as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on alphaIIbbeta3-mediated outside-in signalling, through association with Pyk2.

Since blood platelets release sphingosine 1-phosphate (Sph-1-P) upon activation, it is important to examine the effects of this bioactive lipid on vascular endothelial cell functions from the viewpoint of platelet-endothelial cell interactions. In the present study, we examined Sph-1-P-stimulated signaling pathways related to human umbilical vein endothelial cell (HUVEC) motility, with a special emphasis on the cytoskeletal docking protein Crk-associated substrate (Cas). Sph-1-P stimulated tyrosine phosphorylation of Cas, which was inhibited by the Giinactivator pertussis toxin but not by the Rho inactivator C3 exoenzyme or the Rho kinase inhibitor Y-27632. Fyn constitutively associated with and phosphorylated Cas, suggesting that Cas tyrosine phosphorylation may be catalyzed by Fyn. Furthermore, upon HUVEC stimulation with Sph-1-P, Crk, through its SH2 domain, interacted with tyrosine-phosphorylated Cas, and the Cas-Crk complex translocated to the cell periphery (membrane ruffles), through mediation of Gi (Fyn) but not Rho. In contrast, tyrosine phosphorylation of focal adhesion kinase, and formation of stress fibers and focal adhesion were mediated by Rho but not Gi(Fyn). Finally, Sph-1-P-enhanced HUVEC motility, assessed by a phagokinetic assay using gold sol-coated plates and a Boyden's chamber assay, was markedly inhibited not only by pertussis toxin (or the Fyn kinase inhibitor PP2) but also by C3 exoenzyme (or Y-27632). In HUVECs stimulated with Sph-1-P, these data suggest the following: (i) cytoskeletal signalings may be separable into Gi-mediated signaling pathways (involving Cas) and Rho-mediated ones (involving FAK), and (ii) coordinated signalings from both pathways are required for Sph-1-P-enhanced HUVEC motility. Since HUVECs reportedly express the Sph-1-P receptors EDG-1 (coupled with Gi) and EDG-3 (coupled with G13 and Gq) and the EDG-3 antagonist suramin was found to block specifically Rho-mediated responses, it is likely that Cas-related responses following Gi activation originate from EDG-1, whereas Rho-related responses originate from EDG-3.

Although glycoprotein Ia/IIa (GPIa/IIa, integrin alpha(2)beta(1)) has established its role as a collagen receptor, it remains unclear whether GPIa/IIa mediates activation signals. In this study, we show that rhodocytin, purified from the Calloselasma rhodostoma venom, induces platelet aggregation, which can be blocked by anti-GPIa monoclonal antibodies. Studies with rhodocytin-coupled beads and liposomes loaded with recombinant GPIa/IIa demonstrated that rhodocytin directly binds to GPIa/IIa independently of divalent cations. In vitro kinase assays and Western blotting of GPIa immunoprecipitates revealed that Src and Lyn constitutively associate with GPIa/IIa and that Src activity increases transiently after rhodocytin stimulation. Src specifically associates with p130 Crk-associated substrate (Cas) in a manner dependent upon Cas phosphorylation, suggesting that Src is responsible for Cas tyrosine phosphorylation. While all these phenomena occur early after rhodocytin stimulation in a cAMP-resistant manner, tyrosine phosphorylation of Syk and phospholipase Cgamma2, intracellular Ca(2+) mobilization, and platelet aggregation occur later in a cAMP-sensitive manner. Cytochalasin D, which interferes with actin polymerization and blocks receptor clustering, inhibits all the rhodocytin-mediated signals we examined in this study. We suggest that rhodocytin, by clustering GPIa/IIa, activates GPIa/IIa-associated Src, which then mediates downstream activation signals.

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [32P]Sph-1-P, but not [32P]LPA, was observed in [32P]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca++ mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets, which may be involved in angiogenesis. We, hence, investigated Sph-1-P effects on human umbilical vein endothelial cells (HUVECs) from a viewpoint of angiogenesis. Sph-1-P facilitated HUVEC spreading on the basement membrane component Matrigel, at concentrations ranging from 10 to 250 nM. This stimulatory response induced by Sph-1-P was blocked by pertussis toxin and C3 transferase (from Clostridium botulinum), which inactivate Gi-type heterotrimeric G protein and Rho, respectively. Furthermore, Sph-1-P, in the modified Boyden's chamber assay, stimulated HUVEC migration in a concentration-dependent manner, up to 250 nM. Checkerboard analysis revealed that Sph-1-P markedly induces directional migration (chemotaxis), but a random motility (chemokinesis) was also enhanced. The stimulatory effect of Sph-1-P on HUVEC migration was much stronger than that of other bioactive lipids, and again inhibited by pertussis toxin and by C3 transferase. Our present results that Sph-1-P induces endothelial spreading and migration through Gi-coupled cell surface receptor(s) and Rho are consistent with a recent report on the role of this platelet-derived sphingolipid as a novel regulator of angiogenesis.

Since sphingosine 1-phosphate (Sph-1-P) is stored in abundant amounts in blood platelets and released extracellularly upon stimulation, it is important to clarify the effects of this bioactive lysophospholipid on vascular endothelial cells from the viewpoint of platelet-endothelial cell interactions. In this study, we investigated the effects of Sph-1-P on the cytoskeletal remodeling of human umbilical vein endothelial cells (HUVECs). Of a focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases, HUVECs were found to express FAK, but scarcely proline-rich tyrosine kinase 2. Sph-1-P induced FAK tyrosine phosphorylation, myosin light chain phosphorylation, and the formation of stress fibers in HUVECs. The specific Rho inactivator C3 transferase from Clostridium botulinum abolished all of these cytoskeletal responses induced by Sph-1-P, while pertussis toxin only partly inhibited FAK tyrosine phosphorylation, and hardly affected myosin light chain phosphorylation and stress fiber formation. In contrast, Sph-1-P-induced intracellular Ca(2)(+) mobilization was suppressed by pertussis toxin, but not at all by C3 exoenzyme. Our results suggest that Sph-1-P, a bioactive lipid released from activated platelets, induces endothelial cell cytoskeletal reorganization, mainly through Rho-mediated signaling pathways.

The newly described adapter molecule p130 Crk-associated substrate (Cas) has been reported to contribute to cytoskeletal organization through assembly of actin filaments and to be pivotal in embryonic development and in oncogene-mediated transformation. We characterized the regulation of Cas tyrosine phosphorylation in highly differentiated, anucleate platelets. Phospholipase C-activating receptor agonists, including collagen, thrombin receptor-activating peptide (TRAP), and U46619 (a thromboxane A2 analogue), and A23187 (a Ca2+ ionophore) induced rapid Cas tyrosine phosphorylation in platelets. 12-O-Tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-sn-glycerol, protein kinase C (PKC) activators, also induced Cas tyrosine phosphorylation, albeit sluggishly. Cas tyrosine phosphorylation induced by collagen or TRAP was transient in aggregating platelets; Cas became dephosphorylated in a manner dependent on integrin alpha IIb beta 3-mediated aggregation. While BAPTA-AM (an intracellular Ca2+ chelator) inhibited Cas phosphorylation induced by collagen or TRAP, Ro31-8220 (a PKC inhibitor) rather prolonged it. Under the conditions, this PKC inhibitor suppressed platelet aggregation but not intracellular Ca2+ mobilization. In contrast to Cas involvement in focal adhesions in other cells, platelet Cas phosphorylation preceded the activation of focal adhesion kinase (FAK), and blockage of alpha IIb beta 3-mediated platelet aggregation with a GRGDS peptide resulted in prolongation of stimulation-dependent Cas tyrosine phosphorylation but in suppression of FAK tyrosine phosphorylation. Furthermore, TRAP-induced Cas phosphorylation was insensitive to cytochalasin D, an actin polymerization inhibitor. The failure of FAK to associate with Cas in immunoprecipitation studies also suggests that Cas tyrosine phosphorylation is independent of FAK activation. Of the signaling molecules investigated in this study, Src seemed to associate with Cas. Finally, Cas existed mainly in cytosol and membrane cytoskeleton fractions in the resting state, and remained unchanged during platelet aggregation, when FAK translocated to the cytoskeletal fraction. Our findings on platelet Cas suggest that (i) rapid Cas tyrosine phosphorylation occurs following phosphoinositide turnover by receptor-mediated agonists and may be mediated by intracellular Ca2+ mobilization; (ii) PKC activation, by itself, may elicit sluggish Cas phosphorylation; (iii) Cas tyrosine dephosphorylation, but not phosphorylation, is dependent on integrin alpha IIb beta 3-mediated aggregation; and (iv) Cas is not involved in cytoskeletal reorganization. Anucleate platelets seem to provide a unique model system to fully elucidate the functional role(s) of Cas.

We examined the sphingolipid metabolism of peripheral blood cells, i. e. platelets, erythrocytes, neutrophils and mononuclear cells. A distinguishing characteristic of sphingolipid metabolism in these highly differentiated cells was their high sphingosine (Sph) kinase activity. The occurrence of [3H]sphingosine 1-phosphate (Sph-1-P) from [3H]Sph (actively incorporated from the outside) in the blood cells was strong, long-lasting, and independent of cell activation. Hence, the possibility of Sph-1-P playing a second messenger role is remote in these cells. About 40% of platelet Sph-1-P could be released extracellularly by 12-O-tetradecanoylphorbol 13-acetate, possibly through mediation by protein kinase C. On the other hand, in erythrocytes, neutrophils and mononuclear cells a significant percentage of Sph-1-P formed inside the cell was discharged without stimulation, whereas the stimulation-dependent release was marginal. In contrast to active [3H]Sph conversion to [3H]Sph-1-P, formation of [3H]sphingomyelin was barely detectable in the blood cells; this was especially true for anucleate platelets and erythrocytes. The Sph --> Sph-1-P pathway may become predominant over the Sph --> Cer --> sphingomyelin pathway during late-stage differentiation into platelets or erythrocytes. Sph and its methylated derivative, N, N-dimethylsphingosine, induced apoptosis not only in neutrophils but also in mononuclear cells, whereas Sph-1-P elicited Ca2+ mobilization in platelets. Our results suggest that all blood cells may remove plasma Sph, which is harmful or suppressive to cellular functions, and change it into Sph-1-P, acting as the source of plasma Sph-1-P, which may play a variety of important roles in blood vessels.

Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N,N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [3H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [3H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [3H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca2+ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca2+ mobilization, in platelets.

We evaluated the inhibitory effects of beraprost, a stable prostacyclin analogue, on platelet aggregation, assessed by two methods of platelet aggregometry. The conventional aggregometry detects changes in light transmission (LT) of a platelet suspension, and a recently developed aggregometry based upon a particle counting principle detects light scattering (LS) generated by platelet aggregates. Since LS is more sensitive than LT in detecting platelet aggregates of small size, the minimal concentrations of agonists (ADP, epinephrine, collagen, and U46619) to induce detectable aggregate formation were consistently lower with LS (1/2 to 1/6) than with LT. The effects of beraprost were evaluated on platelet aggregation induced by the optimal concentrations of agonists thus determined for each sample. The IC 50 values of beraprost on platelet aggregation, as assessed by LS, were 1/2 to 1/10 of those assessed by LT. In suppressing platelet aggregation assessed by LS, beraprost was especially potent with IC 50 of 0.2–0.5 nM when platelets were activated by U46619, a thromboxane A 2 analogue, or low concentrations of collagen which activates platelets through thromboxane A 2 production. The IC 50 values were 2–5 nM with ADP and epinephrine, which induce the formation of small aggregates independently of thromboxane A 2 production. These findings suggest that LS can detect inhibitory effects of lower concentrations of antiplatelet agents, since it detects the formation of small aggregates induced by agonists in the lower concentration range than LT. It is also suggested that beraprost potently inhibits thromboxane A 2 -elicited initial signal transduction pathway, reflected by the formation of small aggregates.

von Willebrand factor (vWF ) in the presence of botrocetin induces p72syk activation, assessed as its autophosphorylated level and in vitro kinase assays, the transient association of p72syk with p60c-src, and the translocation of p60c-src and p54/58lyn to cytoskeletal fractions. Jararaca glycoprotein Ib-binding protein (GPIb-BP), which specifically binds to GPIb, abolished these phenomena, suggesting that they are mediated by the vWF-GPIb interaction. These tyrosine kinase-related events were not inhibited by GRGDS peptide (plus EGTA), indicating that GPIIb/IIIa is not involved in the observed responses. Shc, an adaptor protein, was also tyrosine phosphorylated by the botrocetin-vWF activation. When GPIb was immunoprecipitated with nonfunctional monoclonal antibodies (MoAbs) directed against GPIb, a kinase activity was found to associate with GPIb upon botrocetin-vWF activation. On the other hand, anti-GPIb MoAbs that inhibit the vWF-GPIb interaction did not coprecipitate a kinase activity. Because the recovery of GPIb did not differ significantly, it is suggested that the excessive presence of inhibitory anti-GPIb MoAb dissociated a kinase activity from GPIb. Phosphoamino acid analysis showed that the kinase activity was that of a tyrosine kinase. The identity of the tyrosine kinase and the mode of interaction with the cytoplasmic region of GPIb await to be determined. Our findings suggest that the tyrosine kinase associated with GPIb serves at a most proximal step in the signal transduction pathway involved in the vWF-GPIb-induced platelet activation, which leads to other tyrosine kinase-related intracellular signals.

Metabolism of sphingosine (Sph) derivatives in human platelets was examined. [ 3 H]Sph was rapidly and heavily phosphorylated into sphingosine 1-phosphate, similarly in resting and stimulated platelets. [ 14 C] N,N -dimethylsphingosine was stable in resting platelets, while it was converted into N,N -dimethylsphingosine 1-phosphate (DMS-1-P), although weakly, in platelets stimulated with thrombin or 12- O -tetradecanoylphorbol 13-acetate. This DMS-1-P formation was inhibited by staurosporine, a potent protein kinase inhibitor. [ 3 H]C 2 -ceramide was unchanged both in resting and stimulated platelets. Our report is the first to describe production of DMS-1-P in a biological system.

Platelets were activated under the infection with H. pylori in human and mice. We investigated the role of VacA, an exotoxin released by H. pylori in this context. Acid-activated VacA, but not heated VacA, induced platelet CD62P expression. However, VacA reacted with none of the alleged VacA receptors present on platelet membranes. We therefore analyzed VacA associated proteins obtained through VacA affinity chromatography, using MALDI-TOF-MS. Multimerin1 was detected in two consecutive experiments, as the binding protein for VacA. Plasmon resonance confirmed their binding, and dot blot analysis revealed that the peptide sequence AA 321-340 of multimerin 1 is the binding site for VacA. In conclusion, we propose a new interaction between multimerin1 and VacA , which may give another insight into H. pylori-induced platelet activations under H. pylori infection.

We investigated the effects on human platelet aggregation of several agents that increase either intracellular cyclic AMP or cyclic GMP, using a platelet aggregometer that allows quantification of the size and number of platelet aggregates. During the initial phase of aggregation induced by epinephrine and ADP, small aggregates consisting of < 100 cells predominated; large aggregates formed later. Prostaglandin I2 (PGI2), which increases intracellular cyclic AMP, suppressed the formation of small as well as large aggregates induced by epinephrine, with ID50 values of 10.7 +/- 2.8 and 3.8 +/- 0.5 nM, respectively. ADP-induced formation of small and large aggregates was also inhibited by PGI2, with similar ID50 values. Dibutyryl cyclic AMP (db cyclic AMP), a cell-permeant form of cyclic AMP, also inhibited small and large aggregate formation induced by epinephrine or ADP, with ID50 values of 420-560 microM for small aggregates and 139-166 microM for large aggregates, respectively. On the other hand, nitroprusside, which increases intracellular cyclic GMP, inhibited only the formation of large aggregates, with an ID50 value of 454 +/- 191 nM for epinephrine-induced activation and of 2.1 +/- 0.6 microM for ADP-induced activation. Nitroprusside at 1 mM did not affect the formation of small aggregates induced by epinephrine, whereas that of large aggregates was completely blocked at 10 microM. 8-Bromo cyclic GMP (8-br cyclic GMP) also inhibited only the formation of large aggregates, with ID50 values of 140-170 microM, but not that of small aggregates induced by epinephrine and ADP. Milrinone, which increases the intracellular level of both cyclic AMP and cyclic GMP, suppressed the formation of small and large aggregates induced by epinephrine and ADP. These findings suggest that cyclic AMP and cyclic GMP differentially modify the size of aggregates formed during epinephrine or ADP activation.

The effects of S2-serotonergic receptor antagonists, ketanserin, MCI-9042, and one of its major metabolite, M-1, were evaluated on human platelet activation induced by serotonin. A newly developed method for detecting particles in suspensions was used to assess serotonin-induced platelet aggregation. Serotonin added to platelets in plasma induced transient formation of small aggregates but not that of large ones. All the three antagonists in a dose-dependent manner suppressed serotonin-induced platelet aggregation. The ID50 values for ketanserin, MCI-9042, and M-1 are 10 nM, 0.6 microM, and 40 nM, respectively. The effects of these antagonists were also evaluated on [Ca+2]i elevation and shape change, the measurement of which does not require the presence of plasma proteins. These antagonists effectively inhibited [Ca+2]i elevation and shape change induced by serotonin. The ID50 value for MCI-9042 was approximately 1/10 for platelet aggregation. These findings suggest that MCI-9042 tightly binds to plasma proteins with resultant reduction in overall potency. The ID50 values obtained in this study are essentially equivalent to those reported for S2-serotonergic receptor binding in rabbit platelets, suggesting that these agents are also potent antagonists serotonin-induced activation of human platelets.

The effects of three phosphatase inhibitors including okadaic acid, calyculin A and tautomycin were evaluated on platelet Ca2+ mobilization. Calyculin A and tautomycin at appropriate concentrations appeared to have a selective inhibitory effect on thrombin-induced Ca2+ influx, but not on [Ca2+]i release from intracellular Ca2+ storage sites. In contrast, pretreatment with okadaic acid at concentrations that effectively lowered Ca2+ influx also suppressed Ca2+ release from intracellular Ca2+ stores. In a system that specifically evaluates the effects of agents on Ca2+ influx induced by the Ca(2+)-depleted state of intracellular Ca2+ storage sites, the three phosphatase inhibitors attenuated Ca2+ influx in a dose dependent manner and showed complete inhibition at appropriate concentrations. These findings suggest that protein phosphorylation/dephosphorylation plays an important role in mediating signals to open Ca2+ channels when Ca2+ depletion in intracellular Ca2+ stores is caused by thrombin. In contrast, Ca2+ influx induced by thapsigargin, a Ca(2+)-ATPase inhibitor, was only partially suppressed by pretreatment with each of the three phosphatase inhibitors. Based on these findings, we suggest that the Ca(2+)-depleted state of intracellular Ca2+ stores by thapsigargin induces the opening of Ca2+ channels via phosphatase inhibitor-insensitive pathways. All the phosphatase inhibitors, at the highest concentrations tested in the present study, only partially inhibited Mn2+ entry induced by thrombin. These findings suggest that there are at least two types of divalent ion channels on platelet plasma membranes and that one of them, that preferentially allows Mn2+ entry, is resistant to the inhibitory effects of phosphatase inhibitors.

The lectin wheat germ agglutinin (WGA) elicited a prompt and sharp increase in intracellular Ca2+ concentration in human platelets. The WGA- induced Ca2+ mobilization was markedly inhibited by a protein kinase inhibitor staurosporine, whereas Ca2+ mobilization by receptor-mediated agonists, including thrombin, platelet-activating factor, and arginine-vasopressin, was not. In contrast, the lectin-induced Ca2+ mobilization was resistant to cyclic AMP inhibition, compared with that induced by receptor-mediated agonists. These findings indicate that the mechanism of intracellular Ca2+ mobilization, or possibly phospholipase C activation, induced by WGA is different from that induced by receptor-mediated agonists in human platelets.

It is widely accepted that antiplatelet therapy is effective for secondary prevention of atherosclerotic vascular diseases. We performed a double-blind, controlled clinical-pharmacological study to investigate the antiplatelet efficacy of sarpogrelate, a selective 5-hydroxytryptamine (5-HT(2A)) receptor antagonist, in patients with ischemic stroke, using a new assessment system employing combinations of 5-HT and epinephrine as agonists.Forty-seven patients with ischemic stroke were randomly assigned to three groups: 15 patients received 25 mg sarpogrelate (group L), 16 patients received 50 mg (group M), and 15 patients received 100 mg (group H) orally, three times daily for 7 days. The effect was expressed as maximum intensity of platelet aggregation on the last day of medication. Two combinations of agonists, 0.5 micromol/l 5-HT plus 3 micromol/l epinephrine, and 1 micromol/l 5-HT plus 3 micromol/l epinephrine, were used to induce platelet aggregation.With both combinations of agonists, sarpogrelate treatment inhibited platelet aggregation dose-dependently (p0.025, Jonckheere test). In multiple-group comparison, the effect in group H was greater than that in group L or M (p0.025, Wilcoxon rank-sum test).Sarpogrelate treatment inhibited platelet aggregation dose-dependently in patients with ischemic stroke, as judged by a new assessment system employing combinations of 5-HT and epinephrine as agonists.

Platelet aggregation measured by the optical density method has been applied for the assessment of platelet functions. However, as the method has to use platelet-rich plasma, it requires centrifugation of blood samples, which takes a considerable period of time. Using whole blood as samples has advantage because there is no pre-treatment of samples before measurement of platelet functions. Additionally, it would be desirable to have a bedside assay that reflects hyper-function of platelets and can monitor inhibitory effects of anti-platelet drugs. Rapid Platelet Function Assay (RPFA) is a qualitative test to aid the detection of platelet dysfunction due to anti-platelet drug ingestion, which uses citrated whole blood for sample in point of care or laboratory settings. The RPFA is a turbidimetric analysis, based on an optical detection system which measures platelet agglutination as an increase in light transmission. The aim of this study is to assess the accuracy and reproducibility of RPFA and to determine whether RPFA can monitor the effects of anti-platelet drugs. During the first 30 minutes after blood collection, Aspirin Reaction Units (ARU) determined with RPFA gradually increased, and reached its plateau after 30 minutes. The ARU values remained almost constant thereafter until 6 hours after blood collection (Fig. 3). These findings suggest that platelet function is unstable immediately after blood collection. Therefore, in this study ARU measurement was performed 60 minutes after blood collection. The reproducibility of ARU is very good both before and after aspirin intake. Seven days after daily uptake aspirin, ARU was decreased as compared with the control (440.8 +/- 39.4 vs. 663.4 +/- 2.4 ARU). RPFA measurement provides rapid information on platelet function that mirrors turbidimetric platelet agglutination and reflects COX1-depedent platelet activity.

Abdominal aortic aneurysm (AAA) volume and intraluminal thrombi were analyzed with respect to the number and function of platelets, blood cells, and coagulation factors. A group of 43 patients who underwent repair of an AAA were enrolled in this study. The maximum diameter and volume of the AAA, and the volume of intraluminal thrombi and lumen were measured by computed tomography with planimetry. The platelet count and platelet function, prothrombin time, activated partial thromboplastin time, fibrinogen, plasminogen, antithrombin 3, fibrin degradation products (FDP), D-dimer, and blood cell counts were measured. Spontaneous platelet aggregation and the FDP, and D-dimer levels were elevated; all other factors remained within the normal range. Intraluminal thrombus volume was strongly correlated with the volume and diameter of the AAA. However, no correlation was observed between the size of the AAA and coagulating factors, including the number and aggregation value of platelets. AAAs are frequently associated with a coagulating disorder. However, its size and thrombus volume are not correlated with coagulation changes. Although an intraluminal thrornbus increases along with fee enlargement of the AAA, the clinical manifestation of bleeding is rarely associated with an AAA. Therefore coagulopathy in patients with an AAA is not fully explained by its morphology.